Chemoenzymatic synthesis of a biotin-labeled glycophosphononapeptide of the c-Myc oncoprotein

Article abstract of DOI:10.1039/b004822o

Glycophosphopeptides that represent characteristic partial sequences of the posttranslationally modified transcriptional activation domain of the c-Myc oncoprotein can be built up efficiently by a combination of enzymatic and classical chemical techniques

Full text of DOI:10.1039/b004822o

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Chemoenzymatic synthesis of a biotin-labeled glycophosphonona-
peptide of the c-Myc oncoprotein
Thomas Kappes-Rotha and Herbert Waldmann*bc
1
a Organische Chemie, Universität Karlsrühe, Karlsrühe, Germany
b Max-Planck-Institut für Molekulare Physiologie, Otto-Hahn-Strasse 11, D-44227 Dortmund,
Germany
c Organische Chemie, Universität Dortmund, Fb. 3, D-44227 Dortmund, Germany.
Fax: ϩ231-133-2499; E-mail: herbert.waldmann@mpi-dortmund.mpg.de
Received (in Cambridge, UK) 14th June 2000, Accepted 22nd June, 2000
Published on the Web 10th July 2000
Glycophosphopeptides that represent characteristic
partial sequences of the posttranslationally modified tran-
scriptional activation domain of the c-Myc oncoprotein
can be built up efficiently by a combination of enzymatic
and classical chemical techniques.
increased tumourigenicity, suggesting that posttranslational
modification at Thr-58 leads to negative regulation of growth
and neoplastic phenotype.
Covalent modification of serine and threonine residues by
phosphate and O-GlcNAc are highly dynamic processes which
often are reciprocal to each other.6 Therefore, currently it is
unclear which of these modifications at Thr-58 of c-Myc are
beneficial for preventing the oncogenic properties of this pro-
tein. It has, however, been suggested that the form of c-Myc
that interacts with tumour suppressors like the retinoblastoma
protein (Rb) or the p53 protein is the O-GlcNAc-modified
form.6
c-Myc is a nuclear transcription factor that plays a critical role
in the regulation of gene transcription in cell proliferation,
cell differentiation and programmed cell death in normal and
neoplastic cells.1,2 For neoplastic transformation,3a inhibition of
cellular differentiation,3b and induction of apoptosis3c mediated
by c-Myc, the N-terminal transcriptional activation domain
(TAD; see Scheme 1) of the protein is required. Phosphoryl-
ation at Thr-58 and/or Ser-62 in the TAD of c-Myc has been
suggested to modulate the transactivation4a and cellular trans-
formation by Myc.4b Recently it was also shown that Thr-58 is
the major site of covalent attachment of N-acetylglucosamine
to the c-Myc oncoprotein5 (Scheme 1). Furthermore, Thr-58 is
the most important ‘hotspot’ in mutations in c-Myc found in
different types of tumours. This mutation is associated with
In order to study the biological phenomena associated with
covalent modification of c-Myc, characteristic phosphorylated
and glycosylated peptides may be useful reagents. We now
report that c-Myc glycophosphopeptides can be built up effi-
ciently by combination of enzymatic and classical chemical
techniques.
The nonapeptide 1 was chosen as biologically relevant target
compound that represents the correctly glycosylated and phos-
phorylated partial sequence present in the form of Myc that
interacts with the Rb tumour suppressor (vide supra).
In the construction of the peptide conjugate 1, the pro-
nounced acid- and base-lability of glycosylated and phos-
phorylated peptides had to be taken into account. Thus both
glyco- and phosphopeptides undergo a facile β-elimination of
the entire carbohydrate and phosphate even under weakly basic
conditions,7,8 and at acidic pH an anomerization or rupture of
O-glycosidic bonds can occur.8 Therefore, a set of blocking
functions is required that can be removed under the mildest
(preferably neutral) conditions but are orthogonally stable.
To meet these demands we employed the enzyme-labile
p-phenylacetoxybenzyloxycarbonyl group as the temporary
N-terminal protecting group,9 and the Pd0-sensitive allyl
ester10 as the C-terminal blocking function. The carbohydrate
was O-acetylated to guarantee enhanced acid-stability of the
O-glycosidic bond,9,11 and the phosphate was masked with
tert-butyl groups.12
PhAcOZ-protected dipeptide allyl ester 213 was O-phos-
phorylated by treatment with phosphitylating reagent 3 and
subsequent oxidation (Scheme 2). The phosphopeptide 4 there-
by obtained then was selectively deprotected at the C-terminus
by Pd0-mediated transfer of the allyl group to morpholine as
the accepting nucleophile. Coupling of the resulting carboxylic
acid 5 with the N-terminally unmasked dipeptide tert-butyl
ester 6 delivered the fully protected phosphotetrapeptide 7.
From this intermediate the enzyme-labile PhAcOZ urethane
was cleaved off under neutral conditions. In the enzymatic
transformation the biocatalyst recognizes and cleaves the
phenylacetate incorporated into the blocking group. Thus a
phenolate is liberated which undergoes a spontaneous non-
enzymatic fragmentation to give a quinomethane, CO2 and the
desired N-terminally deprotected peptide. The quinomethane is
Scheme 1 Structure of the c-Myc protein. TAD = transcriptional
activation domain; NLS = nuclear localization sequence; bHLH =
basic helix-loop-helix domain; ZIP = leucine zipper domain.
DOI: 10.1039/b004822o
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This journal is © The Royal Society of Chemistry 2000
2579

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The selectively deprotected glycotripeptide 13 and phospho-
tetrapeptide 8 were then coupled to give the complex glyco-
phosphopeptide 14 in high yield (Scheme 4). Peptide conjugate
14 embodies an acid- and base-labile carbohydrate and a base-
labile phosphate group. Nevertheless the N-terminal PhAcOZ
group was removed from this sensitive multifunctional
compound without any undesired side reaction by penicillin
G acylase initiated fragmentation of the urethane. Once
more the substrate specificity of the biocatalyst guarantees that
all other functional groups present are left intact. The reaction
conditions again are so mild that neither β-elimination
nor anomerization occur. By means of this enzymatic trans-
formation the desired selectively deprotected glycophospho-
heptapeptide 15 was obtained in high yield (Scheme 4). After
Scheme 2 Synthesis of the selectively deprotected phosphopeptide
fragment 8.
readily trapped by water or NaHSO3 as nucleophile. In both
the Pd0-mediated removal of the allyl ester and the enzymatic
cleavage of the PhAcOZ urethane no undesired side reaction
was observed. The reaction conditions are so mild that β-
elimination of the phosphate does not occur. Also, the sub-
strate specificity of the biocatalyst guarantees that exclusively
the phenylacetic acid ester is cleaved and that the C-terminal
ester group, the phosphate and the peptide bonds are left intact.
Next, the PhAcOZ-protected threonine derivative 10 was
synthesized from oxazoline 914 and PhAcOZ-Thr and then
condensed with peptide allyl ester 11 to give the completely
protected glycotripeptide 12 in high yield (Scheme 3). From 12,
the C-terminal allyl ester was removed by Pd0-mediated allyl
transfer to morpholine to give carboxylic acid 13.
Scheme 4 Synthesis of the c-Myc glycophosphononapeptide 1 and the
biotin labeled analogue 18.
Scheme 3 Synthesis of the glycopeptide fragment of 13.
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further elongation of the peptide chain by Boc-protected
dipeptide 16,13 all protecting groups were removed from the
glycosylated and phosphorylated nonapeptide 17. To this end,
first all acid-labile tert-butyl groups were cleaved off in one step
by means of trifluoroacetic acid and the O-acetates were
saponified selectively by treatment with hydrazine hydrate in
methanol to give target compound 1 (Scheme 4).
Finally, the glycophosphononapeptide 1 was equipped with a
biotin label by selective N-acylation with biotinylaminocaproic
acid N-hydroxysuccinimide (Biotin-ACA-NHS; Scheme 4). The
biotinylated peptide conjugate 18 obtained in this way may
serve as an efficient molecular probe. The biotin label can be
traced by means of the protein streptavidin which is available
in fluorescently labeled form or modified with colloidal gold
thus allowing the study of a glycosylated and phosphorylated
model protein e.g. by fluorescence microscopy and electron
microscopy in eukaryotic cells.15
In conclusion we have devised a method for the synthesis
of glycosylated and phosphorylated c-Myc peptides by com-
bination of enzymatic and classical chemical transformations.
By means of this methodology biologically relevant labeled
peptide conjugates can be built up which may open up new
avenues of research in biology and bioorganic chemistry.16 In
particular, they should serve to unravel the chemical biology of
the c-Myc protein and the importance of its posttranslational
modification in molecular detail.
Acknowledgements
This research was supported by the Deutsche Forschungs-
gemeinschaft and the Fonds der Chemischen Industrie.
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5 and glycopeptide 13
To a solution of the peptide allyl ester (0.5 mmol) and 2 mol%
of (Ph3P)4Pd in 20 mL of THF under an argon atmosphere was
added dropwise a solution of morpholine (53 µL, 0.6 mmol) in
5 mL of THF. The mixture was stirred for 30 min, the solvent
was removed under reduced pressure and the residue purified
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6 was
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Ser(tBu)-OtBu in 95% yield; this dipeptide was obtained in
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presence of DIC and HOBt as condensation reagents. C-Terminally
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of the benzyl ester from Boc-Leu-Pro-OBzl.
Enzymatic removal of the PhAcOZ group from phosphopeptide 7
and glycophosphopeptide 14
To a solution of the respective PhAcOZ-protected peptide (0.02
mmol) in 8 mL of MeOH, 32 mL of Na2HPO4 buffer was
added, which contained 50 mM NaHSO3 and was adjusted to
pH 7.2 by addition of 0.02 M NaOH. This solution was treated
with penicillin G acylase (160 units) for 4–12 h at room
temperature. The immobilized enzyme was filtered, and the
MeOH was removed under reduced pressure. After lyophilis-
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